Journal of Assisted Reproduction and Genetics

, Volume 33, Issue 9, pp 1231–1238 | Cite as

Reduced sperm DNA longevity is associated with an increased incidence of still born; evidence from a multi-ovulating sequential artificial insemination animal model

  • Stephen D. JohnstonEmail author
  • Carmen López-Fernández
  • Francisca Arroyo
  • Altea Gosálbez
  • Elva I. Cortés Gutiérrez
  • Jose-Luis Fernández
  • Jaime Gosálvez
Reproductive Physiology and Disease



Using a rabbit model, we assessed the influence of sperm DNA longevity on female reproductive outcomes.


Semen was collected from 40 bucks, incubated at 38 °C for 24 h, and the rate of sperm DNA fragmentation (rSDF) was determined using the sperm chromatin dispersion assay. Males were allocated into high rSDF (>0.5 units of increase per hour) or low rSDF (<0.5 units of increase per hour) groups. High and low rSDF semen samples were sequentially artificially inseminated into the same doe to reduce female factor variability, and pregnancy outcomes were recorded.


While there was no difference in SDFs between rSDF groups immediately after collection (T0), differences were significant after 2 h of incubation; SDFs determined at collection and rSDF behaved as independent characters (Pearson correlation = 0.099; P = 0.542). Following artificial insemination, the rate of stillborn pups was significantly higher in does inseminated by males with a high rSDF (14/21) compared to those with low rSDF (15/6); (contingency χ2 5.19; p = 0.022). The risk of stillborn when low rSDF rabbits were used for insemination was 0.16, but increased to 0.36 when high rSDF animals were used (odds ratio = 2.85; 95 % confidence interval = 1.4–2.7).


Dynamic assessment of SDF coupled with natural multiple ovulation, high fecundity of the rabbit and control over female factor influence, provided a useful experimental model to demonstrate the adverse effect of reduced sperm DNA longevity on reproductive outcome.


Oryctolagus cuniculus Sperm DNA fragmentation Still born Sperm DNA longevity Dynamic assay Animal model 



This research was supported by the Spanish Ministry of Economy and Competitiveness, MINECO (BFU-2013-44290-R).


  1. 1.
    Drobnis EZ, Johnson MH. Are we ready to incorporate sperm DNA-fragmentation testing into our male infertility work-up? A plea for more robust studies. Reprod Biomed Online. 2015;30:111–2.Google Scholar
  2. 2.
    Palermo GD, Neri QV, Cozzubbo T, Rosenwaks Z. Perspectives on the assessment of human sperm chromatin integrity. Fertil Steril. 2014;102:1508–17.CrossRefPubMedGoogle Scholar
  3. 3.
    Robinson L, Gallos ID, Conner SJ, Rajkhowa M, Miller D, Lewis S, et al. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Hum Reprod. 2012;27:2908–17.CrossRefPubMedGoogle Scholar
  4. 4.
    Zini A. Are sperm chromatin and DNA defects relevant in the clinic? Syst Biol Reprod Med. 2011;57:78–85.CrossRefPubMedGoogle Scholar
  5. 5.
    Santiso R, Tamayo M, Gosálvez J, Johnston S, Mariño A, Fernández C, et al. DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide. Mutat Res. 2012;734:41–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Practice Committee of the American Society for Reproductive Medicine. The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril. 2013;99:673–7.CrossRefGoogle Scholar
  7. 7.
    Oliva R. Protamines and male infertility. Hum Reprod. 2006;12:417–35.CrossRefGoogle Scholar
  8. 8.
    Gosálvez J, López-Fernández C, Fernández JL, Gouraud A, Holt WV. Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian sperm from eleven species. Mol Reprod Dev. 2011;78:951–61.Google Scholar
  9. 9.
    Gosálvez J, Holt WV, Johnston SD. Sperm DNA fragmentation and its role in wildlife conservation. Reproductive sciences in animal conservation. Adv Exp Med Biol. 2014;753:357–84.CrossRefPubMedGoogle Scholar
  10. 10.
    Gosálvez J, López-Fernández C, Arroyo F, Gosálbez A, Gutiérrez-Cortés El, Johnston SD. The assessment of sperm DNA damage in rabbits using the Halomax assay. In: Adamo G, Constanza A, editors. Rabbits: biology, diet and eating habits and disorders. New York: NOVA Science publishers; 2013. p. 87–100.Google Scholar
  11. 11.
    Bencheik N. Effect de la fréquence de collecte de la semence sur les caractéristiques du sperme et des spermatozoides récoltés chez la lapin. Ann Zootech. 1995;44:263–79.CrossRefGoogle Scholar
  12. 12.
    Mocé E, Vicente JS, Lavara R. Effect of donor strain and maturation stage of rabbit oocytes on results of penetration test of rabbit semen. World Rabbit Sci. 2002;10:53–62.Google Scholar
  13. 13.
    Gosálvez J, López-Fernández C, Fernández JL, Esteves SC, Johnston SD. Unpacking the mysteries of sperm DNA fragmentation: ten frequently asked questions. J Reprod Biotech Fertil. 2015;4:1–16.Google Scholar
  14. 14.
    Aurich C. Recent advances in cooled-semen technology. Anim Reprod Sci. 2008;107:268–75.CrossRefPubMedGoogle Scholar
  15. 15.
    López-Fernández C, Gage MJ, Arroyo F, Gosálbez A, Larrán AM, Fernández JL, et al. Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test. Reproduction. 2009;138:257–66.CrossRefPubMedGoogle Scholar
  16. 16.
    Wdowiak A, Bojar I. Relationship between pregnancy, embryo development, and sperm deoxyribonucleic acid fragmentation dynamics. Saudi J Biol Sci. 2015 (in press). Published online ahead of print 10 August 2015. doi: 10.1016/j.sjbs.2015.08.001.
  17. 17.
    Zhao J, Zhang Q, Wang Y, Li Y. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis. Fertil Steril. 2014;102:998–1005.CrossRefPubMedGoogle Scholar
  18. 18.
    Leach M, Aitken RJ, Sacks G. Sperm DNA fragmentation abnormalities in men from couples with a history of recurrent miscarriage. Aust NZ J Obst Gyn. 2015;55:379–83.Google Scholar
  19. 19.
    Gosálvez J, Caballero P, López-Fernández C, Ortega L, Guijarro JA, Fernández JL, et al. Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors? Asian J Androl. 2013;15:812–8.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Genescà A, Caballín MR, Miró R, Benet J, Germà JR, Egozcue J. Repair of human sperm chromosome aberrations in the hamster egg. Hum Genet. 1992;89:181–6.Google Scholar
  21. 21.
    Derijck A, van der Heijden G, Giele M, Philippens M, de Boer P. DNA double-strand break repair in parental chromatin of mouse zygotes, the first cell cycle as an origin of de novo mutation. Hum Mol Genet. 2008;17:1922–37.Google Scholar
  22. 22.
    Marchetti F, Bishop J, Gingerich J, Wyrobek AJ. Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair. Sci Rep. 2015;5:7689.Google Scholar
  23. 23.
    Gosálvez J, López-Fernández C, Hermoso A, Fernández JL, Kjelland ME. Sperm DNA fragmentation in zebrafish (Danio rerio) and its impact on fertility and embryo viability —Implications for fisheries and aquaculture. Aquaculture. 2014;433:173–82.CrossRefGoogle Scholar
  24. 24.
    Pérez-Cerezales S, Martínez-Páramo S, Beirão J, Herráez MP. Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success. Reproduction. 2010;139:989–97.CrossRefPubMedGoogle Scholar
  25. 25.
    Devaux A, Fiat L, Gillet C, Bony S. Reproduction impairment following paternal genotoxin exposure in brown trout (Salmo trutta) and arctic charr (Salvelinus alpinus). Aquat Toxicol. 2011;101:405–11.CrossRefPubMedGoogle Scholar
  26. 26.
    Fernández-Gonzalez R, Moreira PN, Pérez-Crespo M, Sánchez-Martín M, Ramırez MA, Pericuesta E, et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA fragmented sperm on health and behavior of adult offspring. Biol Reprod. 2008;78:761–72.Google Scholar
  27. 27.
    De Rycke M, Liebaers I, Van Steirteghem A. Epigenetic risks related to assisted reproductive technologies. Risk analysis and epigenetic inheritance. Hum Reprod. 2002;17:2487–94.CrossRefPubMedGoogle Scholar
  28. 28.
    Kelly TL, Trasler JM. Reproductive epigenetics. Clin Genet. 2004;65:247–60.CrossRefPubMedGoogle Scholar
  29. 29.
    Urrego R, Rodriguez-Osorio N, Niemann H. Epigenetic disorders and altered gene expression after use of assisted reproductive technologies in domestic cattle. Epigenetics. 2014;9:803–15.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Stephen D. Johnston
    • 1
    Email author
  • Carmen López-Fernández
    • 2
  • Francisca Arroyo
    • 2
  • Altea Gosálbez
    • 2
  • Elva I. Cortés Gutiérrez
    • 3
  • Jose-Luis Fernández
    • 4
  • Jaime Gosálvez
    • 2
  1. 1.School of Agriculture and Food ScienceThe University of QueenslandGattonAustralia
  2. 2.Faculty of BiologyAutonomous University of MadridCantoblancoSpain
  3. 3.Department of Genetics, Northeastern Biomedical Research CentreThe Mexican Social Security InstituteMonterreyMexico
  4. 4.Unidad de GenéticaComplejo Hospitalario Universitario A Coruña (CHUAC)-INIBIC and Centro Oncológico de GaliciaLa CoruñaSpain

Personalised recommendations